1. Field of the Invention
The present invention relates to non-radioactive vitamin D compounds and methods to assay for the presence of vitamin D, vitamin D analogs and their metabolites which may be present in milk, blood or other biological fluids. The assay methods employed in this invention may be enzyme linked immunoassays (ELISAs) (with biotin containing compounds) and fluorimetric and chemiluminometric assays (with fluorescein or chemiluminiscence containing compounds).
2. Related Art
It is well-established that cutaneously synthesized vitamin D3, a seco-steroid, undergoes sequential metabolic conversions to 25-hydroxyvitamin D3 (25-OH-D3) in the liver and to 1,25(OH)2D3 in the kidney. 1,25(OH)2D3, the dihydroxylated metabolite of vitamin D3, is the most active form of vitamin D hormone which is intimately involved in calcium and phosphorous homeostasis (Holick, M. F. (1989), xe2x80x9cVitamin D: biosynthesis, metabolism and mode of action.xe2x80x9d In Endocrinology, vol. 2, Degroot et al. (eds.), Saunders, W. B., Philadelphia, pp. 902-926). In addition to vitamin D3 (synthesized in the skin), another chemical form of vitamin D3, called vitamin D2, exists in nature. Vitamin D2 is metabolized to 25-hydroxyvitamin D2 (25-(OH)2-D) and 1,25(OH)2D2 in a manner similar to vitamin D3. Vitamin D2 is obtained primarily from diet and vitamin D supplementation, and can be as little as 5-10%, or as high as 100% of the circulating concentration of 25-OH-D depending on the relative amounts of vitamin D2 present in the diet and cutaneously-produced vitamin D3 by exposure to sunlight (Holick, M. F. et al. (1986) xe2x80x9cCalcium, phosphorus and bone metabolism: calcium regulating hormones,xe2x80x9d in Harrison""s Principles of Internal Medicine, 13th Ed., Braunwald el aL (eds.), McGraw-Hill, New York, pp. 2137-2151). In the following discussion, it may be assumed that vitamin D, 25-OH-D and 125(OH)2D will represent the total pool of vitamin D and its metabolites, unless otherwise mentioned.
Biosynthesis of 25-OH-D and 1,25(OH)2D and their metabolism are regulated by the factors that control mineral and skeletal metabolism (Holick, M. F. (1989)). As a result, the serum 1,25(OH)2D level is an important pathophysiological indicator in several diseases. For example, production of 1,25(OH)2D is strongly influenced by a number of diseases such as acquired or inherited disorders of vitamin D-metabolism, including renal osteodystrophy, certain metabolic bone diseases, sarcoidosis, hypercalcemia associated with chronic granulotomous disorders, and vitamin D-dependent rickets types I and II (Holick, M. F. et al. (1986)).
On the other hand, the circulating concentration of 25-OH-D is considered to be an important indicator of vitamin D status in man (Holick, M. F. (1989); Holick, M. F. et al. (1986)). For example, hypovitaminosis, which results from the insufficient endogenous production of vitamin D in the skin, and insufficient dietary supplementation, and/or inability of the small intestine to absorb adequate amounts of vitamin D from diet, results in hypocalcemia and hypophosphatemia and corresponding secondary hyperparathyroidism (Holick, M. F. et al. (1986)). Vitamin D-deficiency is best determined in a clinical setting by measuring 25-OH-D in blood. When the 25-OH-D concentration is below the low limit of the normal range, the patient is considered to be deficient in vitamin D. Hypovitaminosis D also results in disturbances in mineral metabolism (i.e., rickets and osteomalacia in children and adults, respectively).
Serum 25-OH-D-levels are also found to be lower than normal in intestinal malabsorption syndromes, liver disorders (chronic and acute), and nephrotic syndromes. In osteopenia in the aged, serum 25-OH-D levels are often found to be lower than normal. In cases of vitamin D intoxication, serum 25-OH-D level is found, as expected, to be higher than normal (Holick, M. F. et al. (1986)).
Considerable efforts have been directed towards developing assays for accurately measuring concentrations of 25-OH-D in circulation, given its pathophysiological importance. The 25-OH-D assays have been developed for measuring vitamin D status, especially in the elderly and patients with liver diseases and intestinal disorders.
The most efficient methods for assaying 25-OH-D known to date include different variations of a theme that involves competitive binding between xe2x80x98coldxe2x80x99 and xe2x80x98hotxe2x80x99 (radioactive) 25-OH-D3 with normal or vitamin D-deficient rat serum (rat DBP). A standard curve is set up with measured quantities of 25-OH-D3. An organic extract of a blood sample is added to the assay and concentration of 25-OH-D is determined from the standard curve. Serum-concentration of 25-OH-D is much higher (on the order of 100-1000-fold) than the dihydroxylated metabolites of vitamin D, and hence these metabolites do not interfere with the assay in any significant way. This situation is further aided by higher binding avidity of DBP towards 25-OH-D compared with other dihydroxylated metabolites of vitamin D. Furthermnore, DBP does not discriminate between 25-OH-D2 and 25-OH-D3, and hence the measured concentration of 25-OH-D in serum represents the total concentration of 25-OH-D2 and 25-OH-D3 (Chen et al., J. Nutritional Biochem. 1:315-319 (1990)).
DeLuca, U.S. Pat. No. 4,297,289, discloses vitamin D compounds isotopically labeled at the 6-posifion with deuterium or tritium atoms and the use thereof in vitamin D metabolite analyses.
DeLuca, U.S. Pat. No. 4,816,417, discloses a competitive binding assay for the presence of 1,25(OH)2Dx, where x is 2, 3, 4, 5 and/or 6, in a sample containing vitamin D transport protein. According to this assay, receptor protein which is capable of binding to 1,25(OH)2D and labeled 1,25(OH)2D is added to the sample together with an antibody capable of binding to the receptor protein. One then measures the relative degree of binding of labeled 1,25-(OH)2D to the receptor protein. The 1,25(OH)2D is radiolabeled.
The above-mentioned assays, despite their specificity and efficiency, suffer from a few drawbacks. These assays are time-consuming and costly. The most important problem is, however, the intrinsic use of radioactivity. Radioisotopes are very costly, hazardous to handle and store. Radioactive disposal is also becoming an extremely costly affair.
An HPLC-UV detection method, which largely does not use radioactivity, has also been developed for assaying 25-OH-D (Jones, G., Clin. Chem. 24:287-298 (1978)). This method involves multiple chromatographic separations, and final detection and measurement of peaks corresponding to 25-OH-D2 and 25-OH-D3. Although this method provides one of the most accurate measurement of 25-OH-D in serum, it suffers from two major drawbacks. For example, measurement of 25-OH-D is limited by the detection limit of the UV detector. Therefore, 2 ml of blood is needed for the assay. This volume requirement is a particularly difficult problem for determining 25-OH-D levels in younger children. In addition, the assay procedure is very labor intensive and, therefore, very costly.
A non-radioactive method involving isotope-dilution mass spectrometry has also been developed. In this method, a serum sample is spiked with a synthetic analog of 25-OH-D3 which is labeled with stable H-atoms (i.e., deuterium at C-26(27) (Bjorkhem and Holmberg, Clin. Chim. Acta 68:215-224 (1976)) or C6 and C-19 positions (Ray, R. et al., Steroids 57:142-146 (1992)). The xe2x80x98spikedxe2x80x99 serum samples are processed in the usual fashion, i.e. extraction and partial purification of 25-OH-D fraction (by various chromatographic steps), and subjected to mass spectrometry. Concentration of metabolite in the serum is determined by the relative abundance of a particular xe2x80x9cmolecular fragmentxe2x80x9d (generated from the parent metabolite) compared with that of the labeled fragment. This method, although very accurate, has received little practical application due to the requirement of highly sophisticated and expensive instrumentation. This method is also very time-consuming and costly. Furthermore, this method is specific for either 25-OH-D3 or 25-OH-D2, and hence underestimates the total concentration of circulating 25-OH-D.
In summary, 25-OH-D and 1,25(OH)2D assays, known to date, suffer from various disadvantages, including long turn-around time, inter-assay variabilities, high cost and, in particular, the intrinsic use of radioactivity (in most assays), and are not best-suited for routine clinical assays. Furthermore, some of these assays (i.e., HPLC assay) require a fairly large volume of serum. This is a serious problem particularly in cases of neonates, infants and young children. Hence, there is an urgent need for a non-radioactive and highly sensitive method for assaying 25-OH-D and 1,25(OH)2D in blood which will not be tedious, time-consuming and very expensive. The present invention overcomes these problems by providing a novel non-radioactive method for assaying 25-OH-D and 1,25(OH)2D in blood that is rapid, relatively inexpensive and easy to perform.
The measurement of 1,25(OH)2D is very valuable in determining the etiology of inborn and acquired disorders of 25-OH-D metabolism (Holick (1989)). Therefore, the assay of 1,25(OH)2D is valuable clinically and a new method that is rapid and does not use radioactivity is highly desirable.
A large number of active vitamin D compounds are known which are useful for various therapeutic purposes. See, for example, U.S. Pat. Nos. 5,457,217, 5,414,098, 5,384,313, 5,373,004, 5,371,249, 5,430,196, 5,260,290, 5,393,749, 5,395,830, 5,250,523, 5,247,104, 5,397,775, 5,194,431, 5,281,731, 5,254,538, 5,232,836, 5,185,150, 5,321,018, 5,086,191, 5,036,061, 5,030,772, 5,246,925, 4,973,584, 5,354,744, 4,927,815, 4,857,518, 4,851,401, 4,851,400, 4,847,012, 4,755,329, 4,940,700, 4,619,920, 4,594,192, 4,588,716, 4,564,474, 4,552,698, 4,588,528, 4,719,204, 4,719,205, 4,689,180, 4,505,906, 4,769,181, 4,502,991, 4,481,198, 4,448,726, 4,448,721, 4,428,946, 4,411,833, 4,367,177, 4,336,193, 4,360,472, 4,360,471, 4,307,231, 4,307,025, 4,358,406, 4,305,880, 4,279,826, and 4,248,791.
Vitamin D content in milk is determined by a laborious assay (Holick et al, N. Eng. J. Med 132:1178-81 (1992), Tanner et al., J. Assoc. of Analyt. Chem. 17:607-710 (1988)). Thus, there is a need for a rapid non-radioactive assay for vitamin D in milk, blood, body fluids, foods and animal feed. These non-radioactive assays can be used to detect the presence of therapeutically useful vitamin D compounds.
The present invention relates to a compound having the formula: 
wherein:
R1 is a substituted or unsubstituted alkyl, alkenyl or alkynyl group having 1 to 15 C-atoms which may be substituted by one or more hydroxy, halo, lower alkoxy, oxo, oxime, lower alkanoyloxy, aryloxy, aryl, benzoyl, a C4 lactone, a C4 lactone substituted by a methyl and a hydroxy group, C3-C6 cycloalkyl, or C3-C6 cycloalkyl substituted by hydroxy, lower alkyl, or hydroxyloweralkyl;
R2 is a methyl group and R3 is hydrogen, or
R2 is hydrogen and R3 is a methyl group, or
R2 and R3 are both hydrogen or,
R2 and R3 together are a methylene group (xe2x95x90CH2),
R4 is hydrogen, hydroxy, lower alkoxy or lower alkanoyloxy,
W is oxygen or amino;
X is carbonyl (Cxe2x95x90O) or methylene (CH2);
Y is oxygen, sulfur, amino xe2x80x94C(O)Oxe2x80x94 or xe2x80x94C(O)xe2x80x94NHxe2x80x94;
Z is biotin, a fluorescent group or a chemiluminescent group; and n1 and n2 are independently 1, 2, 3, 4, or 5.
The invention also relates to an assay method for the presence of vitamin D compounds in a sample, the improvement comprising using as the labeled vitamin D compound, a labeled compound of the present invention.
The invention also relates to a solid phase assay method for detecting a vitamin D, its metabolite or analog, comprising
(a) providing a solid phase support having immobilized thereon a protein or antibody which is capable of binding to the labeled compound of the present invention;
(b)contacting said solid phase support with a solution of the labeled compound of the invention for a time sufficient to allow binding of the labeled compound to said protein or antibody;
(c) washing the solid phase support obtained in step (b) for a time sufficient to remove unbound labeled compound;
(d) contacting the solid phase support obtained in step (c) with a liquid sample suspected of obtaining a vitamin D, its metabolite or analog for a time sufficient to effect displacement of the labeled compound from said protein or antibody;
(e) removing the liquid obtained in step (d); and
(f) detecting the presence of the labeled compound in the liquid obtained in step (e);
wherein the amount of labeled compound detected in step (f) is directly proportional to the amount of the vitamin D, its metabolite or analog in said test sample.